The goal of this proposal is to determine the X-ray crystal structures of archaeal RNA polymerase and its complexes with auxiliary protein factors and nucleic acid. The transcription apparatus in Archaea can be described as a simplified version of its eukaryotic RNA polymerase II (Pol II) counterpart, comprising a Pol II-like RNA polymerase and the general transcription factors TBP, TFB, TFE and TFS (eukaryotic TBP, TFIIB, TFIIE1 and TFS orthologs, respectively). Remarkably, the transcription regulators found in archaeal genomes are closely related to bacterial factors. Therefore, elucidating the transcription mechanism in Archaea, which is a mosaic of bacterial and eukaryotic features, would provide a foundation for unifying insights from bacterial, archaeal and eukaryotic systems into basic transcription mechanisms across all three domains of life. We recently reported the first X-ray crystal structure of the archaeal RNA polymerase from Sulfolobus solfataricus at 3.4 E resolution. This represents a major breakthrough in our work and provides the foundation for addressing questions on the transcription mechanism in Archaea using structural biology approaches as described below. 1. Complete the Refinement of the Sulfolobus solfataricus RNA Polymerase Model. Our goal is to increase the resolution of the archaeal RNA polymerase structure from 3.4 E to greater than 2.5 E resolution in order to precisely compare the structures of bacterial, archaeal and eukaryotic RNA polymerases. 2. Elucidate the Mechanism of DNA Opening and Transcription Initiation. The archaeal general transcription factors TFB and TFE form stable complexes with RNA polymerase. TFB plays critical roles in recruiting RNA polymerase to the DNA promoter and transcription initiation. TFE facilitates opening of the double-stranded DNA promoter. To understand the mechanism of DNA opening and transcription initiation, we propose to determine the X-ray crystal structures of the archaeal RNA polymerase in a complex with TFB and with TFE, and the entire RNA polymerase transcription pre- initiation complex and open complex with TBP-TFB-promoter DNA. We will carry out structure-based mutational analysis to elucidate the interaction between RNA polymerase and TFB/TFE as well as RNA polymerase and DNA. Because of the high structure similarity between archaeal RNA polymerase and yeast Pol II, we will investigate the mechanism of transcription start site selection in yeast by using mutational analysis based on the archaeal open complex structure. PUBLIC HEALTH RELEVANCE: Gene expression is fundamental to all cellular organisms and is critical for understanding cell development, maintenance, and disease. The archaeal transcription system is amenable to X-ray crystallographic studies, and moreover, is similar to that in eukaryotic cells. Therefore, the proposed studies will provide a structural framework for analyzing four decades of transcription research on diverse eukaryotic systems, and also provide many useful insights into evolution of multi-subunit polymerases in the three domains of life.